厚壳贻贝whirlin类贝壳基质蛋白的重组表达与功能分析

贝壳历来是生物工程和材料学研究的重要对象。贝壳中的贝壳基质蛋白质在贝壳的形成与发育过程中具有重要的调控作用。Whirlin类蛋白质(Whirlin-like protein,WLP)是一种从厚壳贻贝(Mytilus coruscus)中鉴定的新型贝壳基质蛋白质。序列分析结果显示,该蛋白质含有PDZ(postsynaptic density/Discs large/Zonula occludens)结构域,而该结构域对贝壳生物矿化的影响目前尚无报道。为深入了解WLP在贝壳形成中对碳酸钙晶体的影响,在序列分析基础上,采用密码子优化结合原核重组表达,获得其重组表达产物后,开展了重组WLP对碳酸钙晶体形貌及晶型的影响研究,结晶速度抑制以及碳酸钙晶体结合分析。分析结果表明,重组WLP能诱导文石型碳酸钙晶体的形貌和方解石型碳酸钙晶体的晶型发生改变;同时重组WLP对碳酸钙晶体具有结合作用,且能抑制碳酸钙晶体的结晶速度。上述结果表明,WLP对贝壳的形成及发育具有重要影响,并可能在贝壳肌棱柱层的形成中发挥了重要作用。     

外套膜转录物组分析揭示尿素在贻贝耐受海水酸化中的作用

海洋酸化是目前地球面临的主要环境问题之一,对海洋矿化生物的生存带来严重威胁。贝类是目前海水养殖的主要物种,海洋酸化会对贝壳的生物矿化过程产生抑制,因而对贝类养殖业的发展带来严重影响。前期研究中已发现,尿素对贻贝贝壳的形成具有辅助作用,推测尿素有助于贻贝在海洋酸化背景下的生存。为进一步探究尿素通过何种分子机制对贻贝耐受海洋酸化产生积极影响,以厚壳贻贝外套膜为研究对象,采用转录物组学策略分析了尿素添加对贻贝外套膜组织在酸化海水中的基因表达量变化的影响。结果表明,尿素添加对贻贝外套膜在酸化条件下的转录物组变化具有逆转趋势,可抑制外套膜中受酸化胁迫而激活的细胞自噬、凋亡、以及免疫应激相关通路;同时,也诱导了部分贝壳基质蛋白质表达量的上调,从而有助于维持贻贝在酸化条件下的生物矿化过程。上述研究有助于了解贻贝对海洋酸化的耐受性机制,也为后续贝类养殖业在海洋酸化背景下的健康发展提供了新的思路。     

翡翠贻贝外套膜转录组及贝壳珍珠质层和肌棱柱层蛋白质组分析

贝类贝壳在生物材料学及仿生学研究中占据着重要地位。贝壳基质蛋白质是贝壳中的主要有机质成分,对贝壳的形成以及贝壳的力学性能至关重要。翡翠贻贝(Perna viridis)贝壳主要由肌棱柱层和珍珠质层两种微观结构组成,其结构层次较简单,是研究贝壳基质蛋白质及其与贝壳形成关系的极好材料。为深入研究翡翠贻贝贝壳基质蛋白质的分子组成以及分布特点,首先采用扫描电子显微镜,观察翡翠贻贝贝壳内表面珍珠质层和肌棱柱层的微观结构;采用刮取法获得贝壳内表面珍珠质层和肌棱柱层的粉末;对不同层次的贝壳粉末,利用酸溶法去除碳酸钙成分,所获得的有机质组分通过离心将其分为酸可溶性组分和酸不溶性组分。采用Illumina深度测序技术对翡翠贻贝外套膜组织进行大规模测序和序列组装,在此基础上,采用LC-MS/MS质谱技术结合外套膜转录组数据库搜索,对翡翠贻贝肌棱柱层和珍珠质层贝壳基质蛋白质开展组学分析。扫描电镜观察结果表明,翡翠贻贝贝壳有两种不同形貌结构的层次,其中珍珠质层为片状堆叠结构,而肌棱柱层为柱状结构。翡翠贻贝外套膜转录组测序共计获得 69 859 条Unigene。蛋白质组学鉴定结果表明,翡翠贻贝贝壳中总计鉴定到蛋白质54种,其中38种为肌棱柱层所特有蛋白质,3种珍珠质层特有蛋白质,另有13种在珍珠质层和肌棱柱层均被鉴定到。肌棱柱层特有蛋白质的分子多样性明显强于珍珠质层。上述研究为进一步探讨贝壳不同微观层次的形成机制,以及贝壳基质蛋白质对贝壳不同结构层次的调控作用机制奠定了基础。     

利用蛋白质组学手段鉴定新型贻贝足丝蛋白

贻贝通过足腺分泌特有的足丝并以此粘附于水下各种基质表面.贻贝足丝中富含各种粘附蛋白,其优异的水下粘附性能使其成为开发新型生物粘合剂的候选分子.厚壳贻贝足丝粘附能力强,本文采用尿素及盐酸胍抽提结合二维双向电泳技术（two-dimensional electrophoresis, 2-DE）,分别对厚壳贻贝足丝纤维和足丝盘的蛋白质进行分离及染色;采用串联质谱技术结合常规搜库和表达序列标签（EST) 数据库搜索,对分离获得的蛋白质点进行鉴定,从中获得了mfp-3、mfp-6、胶原蛋白以及3种未曾报道过的新型贻贝足丝蛋白成分.上述研究为深入了解厚壳贻贝足丝蛋白的分子多样性、探讨其粘附机理以及从中筛选具有应用前景的贻贝足丝蛋白奠定了基础.     

厚壳贻贝足丝黏附蛋白mfp-3的重组表达及黏附功能分析

厚壳贻贝（Mytilus coruscus）中富含各种黏附蛋白分子,其中贻贝足丝蛋白3（mussel foot protein-3, mfp-3）是贻贝用以与外界基质进行黏附的主要蛋白分子.贻贝足丝中天然的mfp-3的含量低,水溶性差,因此纯化困难.本文以厚壳贻贝足丝蛋白mfp-3的cDNA序列为目的基因,用PCR法扩增Mfp-3基因,并成功构建含有多聚组氨酸标签的重组mfp-3原核表达载体pET-21a/ Mfp-3.经IPTG（isopropylthio-β-D-galactoside）诱导表达出重组蛋白,利用亲和层析和反相高效液相色谱分离纯化,获得分子量为9.18 kD的重组蛋白.经酪氨酸酶催化、玻璃包被和石英晶体微天平（quartz crystal microbalance,QCM）分析.结果表明,重组厚壳贻贝mfp-3蛋白经酪氨酸酶催化后,L-3,4-二羟基苯丙氨酸(即多巴,L-3,4- dihydroxyphenylalanine, DOPA) 含量较高并且具有较好的黏附性能.上述研究为开发以mfp-3黏附蛋白为来源的生物粘合剂奠定了良好的基础.     

壳损伤及海水酸化对厚壳贻贝脲酶和碳酸酐酶的影响

海洋酸化是当前全球面临的最为紧迫的环境问题之一，已显现出对具生物矿化现象物种的严重影响。以往研究发现，贻贝表现出对海洋酸化较强的耐受性。为探究贻贝对海洋酸化耐受性的可能机制，选择两种对生物矿化具有重要影响的酶(碳酸酐酶和脲酶)为研究对象，分析其在壳损伤以及酸化海水条件下基因表达量和酶活力的变化；进一步对上述条件下的贻贝贝壳内表面开展了显微观察。研究结果表明，相比对照组，壳损伤或酸化海水处理诱导碳酸酐酶和脲酶的基因表达量产生不同程度的上调(P<0.05),酶活力测试与基因表达量分析结果具有类似特征，但存在时序性差异。而壳损伤叠加海水酸化处理则诱导碳酸酐酶和脲酶的基因表达量及酶活性在外套膜中均明显下调(P<0.05),但碳酸酐酶在血细胞中明显上调(P<0.05);在酸化海水中添加尿素则明显上调血细胞和外套膜中碳酸酐酶和脲酶的基因表达量以及酶活性(P<0.05)。贝壳内表面显微观察结果进一步表明，海水酸化及壳损伤导致损伤部位附近的贝壳内表面产生明显纹理质地改变，尿素可诱导海水酸化条件下壳损伤部位修复层的重新出现。上述结果表明，碳酸酐酶和脲酶可能参与了对壳损伤修复及海洋...     

厚壳贻贝鸟氨酸-尿素循环中的关键代谢物及基因分析

鸟氨酸-尿素循环(OUC)是生物新陈代谢过程中的重要循环过程,但在贝类中尚缺乏相关研究。为此,以厚壳贻贝为研究对象,分别采用氨基酸分析仪和荧光定量PCR研究了其外套膜和后闭壳肌组织中的鸟氨酸-尿素循环途径的主要代谢物和关键基因的含量及其表达量;进一步测试了在精氨酸注射条件下,各主要代谢物和关键基因的含量及表达量变化,以及¹³C标记尿素注射贻贝后,其贝壳中δ¹³C比值(¹³C/¹²C)变化。结果表明,厚壳贻贝外套膜和后闭壳肌均含有较高浓度的尿素;精氨酸注射导致其两种组织中尿素浓度显著上升(P<0.01),以及瓜氨酸浓度显著下降(P<0.01),但鸟氨酸浓度维持相对稳定的水平。精氨酸注射显著上调了两种组织中的脲酶基因的表达量(P<0.01),但其他基因表达量的变化在两种组织中存在差异,显示出鸟氨酸-尿素循环途径在其两种组织中具有复杂而不同的调控过程。¹³C标记尿素注射贻贝显著上调了贝壳中δ¹³C的比值(P <0.01),表明尿素分子可能参与了贻贝贝壳的生物矿化过程。上述研究为深入了解贻贝鸟氨酸-尿素途径与生物矿化之间的关联,以及探讨贻贝对海水酸化耐受性的内在分子机制奠定了基础。     

厚壳贻贝足丝盘黏附蛋白mcofp-3的重组真核表达

厚壳贻贝(Mytilus coruscus)黏附蛋白分子mcofp-3(M.coruscusfoot protein-3)主要分布于贻贝足丝盘,贻贝在水环境下的黏附过程中起到关键作用,但因其难溶于水且在贻贝足丝盘中含量极低,故妨碍了对其进行深入研究。为建立厚壳贻贝足丝蛋白mcofp-3的真核表达体系,并获得足够的mcofp-3黏附蛋白进行后续研究,采用酵母表达体系对mcofp-3进行了重组表达。通过PCR方法克隆厚壳贻贝的mcofp-3基因,构建mcofp-3的酵母真核表达载体pVT102U/α/mcofp-3,鉴定结果表明,重组表达质粒pVT102U/α/mcofp-3由真核载体pVT102U/α和mcofp-3的成熟肽DNA片段组成,插入的mcofp-3成熟肽DNA片段与预期序列完全一致;采用LiAC转化法将重组表达质粒转化到S78酿酒酵母中,经过RT-PCR分析以及1.0%的琼脂糖凝胶电泳检测,结果表明,重组的mcofp-3得到了成功的转录;发酵菌液经阳离子交换柱及高效液相色谱分离,以及Tris-Tricine-SDS-PAGE检测,结果表明,重组的厚壳贻贝黏附蛋白分子mcofp-3得到了成功表达,表达...     

基于illumina测序的厚壳贻贝足组织转录组研究

贻贝足丝是贻贝足组织分泌的足丝蛋白形成的非细胞组织,具有在水环境下的极强粘附性能,是当前生物粘附剂及抗腐蚀材料的研发热点．为进一步了解贻贝足丝蛋白的分子多样性特征,采用新一代Illumina高通量测序平台对厚壳贻贝(Mytilus coruscus)足组织进行转录组测序,首次构建了厚壳贻贝足组织的转录组数据库．共计获得7 199 799 840 nt的碱基数据经过序列拼接和组装,获得88 825条unigene．对上述unigene开展了序列注释,共计37 007条unigene获得注释．在此基础上,经序列检索和比对,从中筛选出与目前已知的11种足丝蛋白同源的56条unigene序列并进行分析．结果表明,厚壳贻贝足丝蛋白具有明显的氨基酸偏好性,部分足丝蛋白具有重复序列,且厚壳贻贝足丝蛋白与其他种类的贻贝足丝蛋白具有较高的序列相似性．上述结果为后续贻贝足丝蛋白的批量鉴定以及在此基础上的贻贝足丝形成、固化以及粘附机制相关研究奠定了基础．     

厚壳贻贝低分子量足丝蛋白的初步鉴定(英文)

足丝蛋白是贻贝科(Mytilidae)所特有一种在水环境中也能表现出强黏附功能的蛋白,也是目前开发新型生物黏附剂的主要候选分子。厚壳贻贝(Mytilus coruscus)广泛分布于我国东部沿海,是我国具有重要经济价值的贻贝,其足丝粗硬,黏附力强,关于厚壳贻贝的足丝蛋白的研究目前尚未见报道。通过醋酸抽提结合反相高效液相色谱分离,从厚壳贻贝足丝盘中分离纯化到数种低分子量足丝蛋白,经质谱鉴定和氨基酸序列测定,其中三种足丝蛋白(分子量6 kD左右)属于贻贝足丝蛋白-3(mytilus foot protein-3,mfp-3)家族,且序列中富含DOPA,另有三种足丝蛋白为未知新型足丝蛋白。石英晶体微天平分析表明,厚壳贻贝低分子量足丝蛋白在金表面有较强的吸附能力,这与其黏附功能是直接相关的。以上工作为深入了解厚壳贻贝低分子量足丝蛋白的分子多样性以及黏附机制奠定了基础。     

Identification of genes directly involved in shell formation and their functions in pearl oyster, Pinctada fucata

Mollusk shell formation is a fascinating aspect of biomineralization research. Shell matrix proteins play crucial roles in the control of calcium carbonate crystallization during shell formation in the pearl oyster, Pinctada fucata. Characterization of biomineralization-related genes during larval development could enhance our understanding of shell formation. Genes involved in shell biomineralization were isolated by constructing three suppression subtractive hybridization (SSH) libraries that represented genes expressed at key points during larval shell formation. A total of 2,923 ESTs from these libraries were sequenced and gave 990 unigenes. Unigenes coding for secreted proteins and proteins with tandem-arranged repeat units were screened in the three SSH libraries. A set of sequences coding for genes involved in shell formation was obtained. RT-PCR and in situ hybridization assays were carried out on five genes to investigate their spatial expression in several tissues, especially the mantle tissue. They all showed a different expression pattern from known biomineralization-related genes. Inhibition of the five genes by RNA interference resulted in different defects of the nacreous layer, indicating that they all were involved in aragonite crystallization. Intriguingly, one gene (UD_Cluster94.seq.Singlet1) was restricted to the 'aragonitic line'. The current data has yielded for the first time, to our knowledge, a suite of biomineralization-related genes active during the developmental stages of P. fucata, five of which were responsible for nacreous layer formation. This provides a useful starting point for isolating new genes involved in shell formation. The effects of genes on the formation of the 'aragonitic line', and other areas of the nacreous layer, suggests a different control mechanism for aragonite crystallization initiation from that of mature aragonite growth.     

Ubiquitylation functions in the calcium carbonate biomineralization in the extracellular matrix

Mollusks shell formation is mediated by matrix proteins and many of these proteins have been identified and characterized. However, the mechanisms of protein control remain unknown. Here, we report the ubiquitylation of matrix proteins in the prismatic layer of the pearl oyster, Pinctada fucata. The presence of ubiquitylated proteins in the prismatic layer of the shell was detected with a combination of western blot and immunogold assays. The coupled ubiquitins were separated and identified by Edman degradation and liquid chromatography/mass spectrometry (LC/MS). Antibody injection in vivo resulted in large amounts of calcium carbonate randomly accumulating on the surface of the nacreous layer. These ubiquitylated proteins could bind to specific faces of calcite and aragonite, which are the two main mineral components of the shell. In the in vitro calcium carbonate crystallization assay, they could reduce the rate of calcium carbonate precipitation and induce the calcite formation. Furthermore, when the attached ubiquitins were removed, the functions of the EDTA-soluble matrix of the prismatic layer were changed. Their potency to inhibit precipitation of calcium carbonate was decreased and their influence on the morphology of calcium carbonate crystals was changed. Taken together, ubiquitylation is involved in shell formation. Although the ubiquitylation is supposed to be involved in every aspect of biophysical processes, our work connected the biomineralization-related proteins and the ubiquitylation mechanism in the extracellular matrix for the first time. This would promote our understanding of the shell biomineralization and the ubiquitylation processes.     

Characterization of a novel shell matrix protein with vWA domain from Mytilus coruscus

ABSTRACT

Mollusk shell is a product of biomineralization with excellent mechanical properties, and the shell matrix proteins (SMPs) have important functions in shell formation. A vWA domain-containing protein (VDCP) was identified from the shell of Mytilus coruscus as a novel shell matrix protein. The VDCP gene is expressed at a high level in specific locations in the mantle and adductor muscle. Recombinant VDCP (rVDCP) showed abilities to alter the morphology of both calcite and aragonite, induce the polymorph change of calcite, bind calcite, and decrease the crystallization rate of calcite. In addition, immunohistochemistry analyses revealed the specific location of VDCP in the mantle, the adductor muscle, and the myostracum layer of the shell. Furthermore, a pull-down analysis revealed eight protein interaction partners of VDCP in shell matrices and provided a possible protein–protein interaction network of VDCP in the shell.     

A Novel Acidic Matrix Protein,PfN44, Stabilizes Magnesium Calcite to Inhibit the Crystallization of Aragonite

Magnesium is widely used to control calcium carbonate deposition in the shell of pearl oysters. Matrix proteins in the shell are responsible for nucleation and growth of calcium carbonate crystals. However, there is no direct evidence supporting a connection between matrix proteins and magnesium. Here, we identified a novel acidic matrix protein named PfN44 that affected aragonite formation in the shell of the pearl oyster Pinctada fucata. Using immunogold labeling assays, we found PfN44 in both the nacreous and prismatic layers. In shell repair, PfN44 was repressed, whereas other matrix proteins were up-regulated. Disturbing the function of PfN44 by RNAi led to the deposition of porous nacreous tablets with overgrowth of crystals in the nacreous layer. By in vitro circular dichroism spectra and fluorescence quenching, we found that PfN44 bound to both calcium and magnesium with a stronger affinity for magnesium. During in vitro calcium carbonate crystallization and calcification of amorphous calcium carbonate, PfN44 regulated the magnesium content of crystalline carbonate polymorphs and stabilized magnesium calcite to inhibit aragonite deposition. Taken together, our results suggested that by stabilizing magnesium calcite to inhibit aragonite deposition, PfN44 participated in P. fucata shell formation. These observations extend our understanding of the connections between matrix proteins and magnesium.     

A novel extrapallial fluid protein controls the morphology of nacre lamellae in the pearl oyster, Pinctada fucata

Mollusk shell nacre is known for its superior mechanical properties and precisely controlled biomineralization process. However, the question of how mollusks control the morphology of nacre lamellae remains unresolved. Here, a novel 38-kDa extrapallial fluid (EPF) protein, named amorphous calcium carbonate-binding protein (ACCBP), may partially answer this question. Although sequence analysis indicated ACCBP is a member of the acetylcholine-binding protein family, it is actively involved in the shell mineralization process. In vitro, ACCBP can inhibit the growth of calcite and induce the formation of amorphous calcium carbonate. When ACCBP functions were restrained in vivo, the nacre lamellae grew in a screw-dislocation pattern, and low crystallinity CaCO(3) precipitated from the EPF. Crystal binding experiments further revealed that ACCBP could recognize different CaCO(3) crystal phases and crystal faces. With this capacity, ACCBP could modify the morphology of nacre lamellae by inhibiting the growth of undesired aragonite crystal faces and meanwhile maintain the stability of CaCO(3)-supersaturated body fluid by ceasing the nucleation and growth of calcite. Furthermore, the crystal growth inhibition capacity of ACCBP was proved to be directly related to its acetylcholine-binding site. Our results suggest that a "safeguard mechanism" of undesired crystal growth is necessary for shell microstructure formation.     

Ocean acidification and temperature increase impact mussel shell shape and thickness: problematic for protection?

Ocean acidification threatens organisms that produce calcium carbonate shells by potentially generating an under‐saturated carbonate environment. Resultant reduced calcification and growth, and subsequent dissolution of exoskeletons, would raise concerns over the ability of the shell to provide protection for the marine organism under ocean acidification and increased temperatures. We examined the impact of combined ocean acidification and temperature increase on shell formation of the economically important edible mussel Mytilus edulis. Shell growth and thickness along with a shell thickness index and shape analysis were determined. The ability of M. edulis to produce a functional protective shell after 9 months of experimental culture under ocean acidification and increasing temperatures (380, 550, 750, 1000 μatm pCO ₂, and 750, 1000 μatm pCO ₂ + 2°C) was assessed. Mussel shells grown under ocean acidification conditions displayed significant reductions in shell aragonite thickness, shell thickness index, and changes to shell shape (750, 1000 μatm pCO ₂) compared to those shells grown under ambient conditions (380 μatm pCO ₂). Ocean acidification resulted in rounder, flatter mussel shells with thinner aragonite layers likely to be more vulnerable to fracture under changing environments and predation. The changes in shape presented here could present a compensatory mechanism to enhance protection against predators and changing environments under ocean acidification when mussels are unable to grow thicker shells. Here, we present the first assessment of mussel shell shape to determine implications for functional protection under ocean acidification.     

The interstitial crystal-nucleating sheet in molluscan Haliotis rufescens shell: A bio-polymeric composite

The interstitial green sheets in abalone shell nacre are shown to be bifacially differentiated trilaminate polymeric complexes, with glycoprotein layers sandwiching a central core containing chitin. They share some common feature with the organic matrix layers between the aragonite tablets in the nacre and the periostracum, and show similarities to the myostracum. Thus, although the green sheet is reported to be unique to the abalone shell, it represents an interesting model for the study of molluscan shell biomineralization processes. Indeed, during shell formation, prismatic and spherulitic aragonite precedes and follows the deposition of the interstitial green polymeric composite sheets, and there is evidence to suggest that these sheets demark the interruption of nacre synthesis and serve to nucleate the resumption of calcium carbonate crystal growth. The green polymeric interstitial sheet purified from the abalone shell was investigated by spectroscopic and imaging techniques: FTIR, confocal microscopy, scanning and transmission electron microscopy, and by pyrolysis combined with GC–MS. Structural and compositional differences are observed between the surfaces of the two sides of the interstitial polymeric composite sheets. Moreover, comparative crystallization experiments on the green sheet sides also reveal asymmetry with respect to the nucleation of calcium carbonate. These findings suggest that these bifacially differentiated interstitial composites may play an active role in the mineral assembly processes, with one of the surfaces acting as a crystal nucleator.